EP2548024A2 - Fluoreszierende arsenhaltige mittel und assays damit - Google Patents

Fluoreszierende arsenhaltige mittel und assays damit

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Publication number
EP2548024A2
EP2548024A2 EP11757137A EP11757137A EP2548024A2 EP 2548024 A2 EP2548024 A2 EP 2548024A2 EP 11757137 A EP11757137 A EP 11757137A EP 11757137 A EP11757137 A EP 11757137A EP 2548024 A2 EP2548024 A2 EP 2548024A2
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EP
European Patent Office
Prior art keywords
optionally
optionally substituted
compound
hetero
arsenic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP11757137A
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English (en)
French (fr)
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EP2548024A4 (de
Inventor
Laura Ellen Downs Beaulieu
Mary J. Tanga
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SRI International Inc
Original Assignee
SRI International Inc
Stanford Research Institute
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Publication of EP2548024A2 publication Critical patent/EP2548024A2/de
Publication of EP2548024A4 publication Critical patent/EP2548024A4/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/66—Arsenic compounds
    • C07F9/70—Organo-arsenic compounds
    • C07F9/80—Heterocyclic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/66—Arsenic compounds
    • C07F9/70—Organo-arsenic compounds
    • C07F9/80—Heterocyclic compounds
    • C07F9/88—Arsenic compounds containing one or more acridine ring systems
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B11/00—Diaryl- or thriarylmethane dyes
    • C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
    • C09B11/10—Amino derivatives of triarylmethanes
    • C09B11/24—Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B17/00—Azine dyes
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B19/00—Oxazine dyes
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B21/00—Thiazine dyes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/44—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa

Definitions

  • the field of the invention is arsenical fluorescent agents and diagnostic assays.
  • NTDs Neglected Tropical Diseases
  • Chagas' disease African Trypanosomiasis or Sleeping Sickness
  • Leishmaniasis a group of infections that most commonly plague those who are extremely poor and live in remote rural areas, urban slums, and places of political conflict. Because they adversely affect child development, pregnancy, and worker productivity, NTDs are a major reason why many in developing nations cannot escape extreme poverty. Three such diseases are caused by trypanosomal parasites: Chagas' disease, African Trypanosomiasis or Sleeping Sickness, and Leishmaniasis.
  • the invention provides methods and compositions for labeling dithiol-containing analytes.
  • the invention provides a substituted, optionally hydro-, optionally hetero-, monoarsenical anthracene compound comprising a 4' rotation blocking group and a 5' arsenic, and that exhibits a detectable increase or shift in fluorescence when the arsenic reacts with two thiols of a rotation-blocking binding target molecule, the compound having the structure I:
  • Rl is O, S, NRa, or CRa 2 ;
  • R2 is C or N
  • R3 is optionally substituted-, optionally hetero- alkyl, optionally substituted-, optionally hetero- alkenyl, optionally substituted-, optionally hetero- alkynyl, optionally substituted-, optionally hetero-aryl, or optionally substituted-, optionally hetero- alkoxy;
  • R4 and R5 are independently carbonyl, carbonothioyl, NRa 2 , ORa, or SRa ;
  • R6 and R7 are Ra, halide, ORa, NRa 2 , ORa, SRa, or nitro (-N0 2 );
  • R8 and R9 are Ra, halide, ORa, NRa 2 , ORa, SRa, or nitro (-N0 2 );
  • R10 is a rotational blocking group
  • Rl 1 is an arsenic protecting group displaced by reaction of the arsenic with the thiols of the target molecule;
  • Ra groups are independently H, optionally substituted-, optionally hetero- alkyl, optionally substituted-, optionally hetero- alkenyl, optionally substituted-, optionally hetero- alkynyl, optionally substituted-, optionally hetero-aryl, or optionally substituted-, optionally hetero- alkoxy;
  • Rl l is carbonyl, 1,2 ethanedithiol, or dihydroxyl
  • the rotation blocking group is an optionally substituted-, optionally hetero- alkyl, optionally substituted-, optionally hetero- alkenyl, optionally substituted-, optionally hetero- alkynyl, optionally substituted-, optionally hetero-aryl, or optionally substituted-, optionally hetero- alkoxy
  • the anthracene compound is a fluoresein, rhodamine, eosin, phenazine, phenoxazine, phenothiazine, thioxanthene, acridine, or a core or derivative thereof.
  • the invention provides a substituted, optionally hydro-, optionally hetero-, monoarsenical anthracene compound comprising a 4' rotation- blocking group and a 5' arsenic, and that exhibits a detectable increase or shift in
  • AC is the anthracene core
  • RB is the rotation blocking group
  • TM is the target molecule
  • the invention also provide methods of making and using the subject compounds, including a method of using a subject compound comprising the step of: contacting the compound with the target molecule wherein the arsenic reacts with the thiols, and in particular, a method of diagnosing a trypanasomatid infection in a patient by detecting a dicysteine trypanathione, comprising the steps of: (a) contacting a sample of the patient with a subject compound; and (b) detecting the increase or shift in fluorescence as an indication of the presence of the trypanathione and the trypanasomatid infection.
  • the invention also provides a method of diagnosing a trypanasomatid infection in a patient by detecting a dicysteine trypanathione, comprising the steps of: (a) contacting a sample of the patient with an arsenical fluorescent dye that exhibits a detectable increase or shift in fluorescence when the arsenic of the dye reacts with the cysteines of the
  • trypanathione and (b) detecting the increase or shift in fluorescence as an indication of the presence of the trypanathione and the trypanasomatid infection.
  • the subject monoarsenical dyes have a significant advantage over diarsenical dyes in diagnoses, in that they form a brightly fluorescent complex upon binding of a dicysteine motif, whereas he 1 : 1 complex with prior diarsenical dyes is only weakly fluorescent. This in not a flaw of the diarsenicals, but reflect their distinct design criteria. Biarsenicals are used in excess to track a small concentration of proteins as they are trafficked through cells, and hence requireaki selectivity and high binding affinity afforded by the tetradentate binding capacity of the bisarsenical. Essentially, the biarsenticals were engineered as a small molecule equivalent of GFP to monitor protein trafficking.
  • the invention provides a substituted, optionally hydro-, optionally hetero-, monoarsenical anthracene compound comprising a 4' rotation-blocking group and a 5' arsenic, and that exhibits a detectable increase or shift in fluorescence when the arsenic reacts with two thiols of a rotation-blocking binding target molecule forming a conjugate having the general structure II:
  • AC is the anthracene core
  • RB is the rotation blocking group
  • TM is the target molecule
  • the invention provides a substituted, optionally hydro-, optionally hetero-, monoarsenical anthracene compound comprising a 4' rotation blocking group and a 5' arsenic, and that exhibits a detectable increase or shift in fluorescence when the arsenic reacts with two thiols of a rotation-blocking binding target molecule, the compound having the structure I:
  • Rl is O, S, NRa, or CRa 2 ;
  • R2 is C or N
  • R3 is optionally substituted-, optionally hetero- alkyl, optionally substituted-, optionally hetero- alkenyl, optionally substituted-, optionally hetero- alkynyl, optionally substituted-, optionally hetero-aryl, or optionally substituted-, optionally hetero- alkoxy;
  • R4 and R5 are independently carbonyl, carbonothioyl, NRa 2 , ORa, or SRa ;
  • R6 and R7 are Ra, halide, ORa, NRa 2 , ORa, SRa, or nitro (-N0 2 );
  • R8 and R9 are Ra, halide, ORa, NRa 2 , ORa, SRa, or nitro (-N0 2 );
  • R10 is a rotational blocking group
  • Rl 1 is an arsenic protecting group displaced by reaction of the arsenic with the thiols of the target molecule
  • Ra groups are independently H, optionally substituted-, optionally hetero- alkyl, optionally substituted-, optionally hetero- alkenyl, optionally substituted-, optionally hetero- alkynyl, optionally substituted-, optionally hetero-aryl, or optionally substituted-, optionally hetero- alkoxy;
  • genuses are recited as shorthand for a recitation of all members of the genus; for example, the recitation of (C1-C3) alkyl is shorthand for a recitation of all C1-C3 alkyls: methyl, ethyl and propyl, including isomers thereof.
  • heteroatom as used herein generally means any atom other than carbon, hydrogen or oxygen.
  • Preferred heteroatoms include oxygen (O), phosphorus (P), sulfur (S), nitrogen (N), silicon (S), arsenic (As), selenium (Se), and halogens
  • preferred heteroatom functional groups are haloformyl, hydroxyl, aldehyde, amine, azo, carboxyl, cyanyl, thocyanyl, carbonyl, halo, hydroperoxyl, imine, aldimine, isocyanide, iscyante, nitrate, nitrile, nitrite, nitro, nitroso, phosphate, phosphono, sulfide, sulfonyl, sulfo, and sulfhydryl.
  • alkyl by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which is fully saturated, having the number of carbon atoms designated (i.e. C1-C8 means one to eight carbons).
  • alkyl groups include methyl, ethyl, n-propyl, isopropyl, n- butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl and the like.
  • alkenyl by itself or as part of another substituent, means a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be mono- or polyunsaturated, having the number of carbon atoms designated (i.e. C2-C8 means two to eight carbons) and one or more double bonds.
  • alkenyl groups include vinyl, 2- propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl) and higher homologs and isomers thereof.
  • alkynyl by itself or as part of another substituent, means a straight or branched chain hydrocarbon radical, or combination thereof, which may be mono- or polyunsaturated, having the number of carbon atoms designated (i.e. C2-C8 means two to eight carbons) and one or more triple bonds.
  • alkynyl groups include ethynyl, 1- and 3-propynyl, 3-butynyl and higher homologs and isomers thereof.
  • alkylene by itself or as part of another substituent means a divalent radical derived from alkyl, as exemplified by -CH 2 -CH 2 -CH 2 -CH 2 -.
  • an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the invention.
  • a “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
  • alkoxy alkylamino and “alkylthio” (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively.
  • heteroalkyl by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and from one to three heteroatoms selected from the group consisting of O, N, Si and S, wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
  • the heteroatom(s) O, N and S may be placed at any interior position of the heteroalkyl group.
  • the heteroatom Si may be placed at any position of the heteroalkyl group, including the position at which the alkyl group is attached to the remainder of the molecule.
  • Up to two heteroatoms may be consecutive, such as, for example, -CH 2 -NH-OCH 3 and -CH 2 -0-Si(CH 3 ) 3 .
  • heteroalkylene by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified by -CH 2 -CH 2 -S-CH 2 -CH 2 - and - CH 2 -S-CH 2 -CH 2 -NH-CH 2 -.
  • heteroalkylene groups heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied.
  • cycloalkyl and heterocycloalkyl represent, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl”, respectively. Accordingly, a cycloalkyl group has the number of carbon atoms designated (i.e., C3-C8 means three to eight carbons) and may also have one or two double bonds.
  • a heterocycloalkyl group consists of the number of carbon atoms designated and from one to three heteroatoms selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
  • heterocycloalkyl a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule.
  • cycloalkyl include cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like.
  • heterocycloalkyl include l-(l,2,5,6-tetrahydropyrid- yl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,
  • halo and “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
  • terms such as “haloalkyl,” are meant to include alkyl substituted with halogen atoms, which can be the same or different, in a number ranging from one to (2m'+l), where m' is the total number of carbon atoms in the alkyl group.
  • halo(Cl-C4)alkyl is mean to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
  • haloalkyl includes monohaloalkyl (alkyl substituted with one halogen atom) and polyhaloalkyl (alkyl substituted with halogen atoms in a number ranging from two to (2m'+l) halogen atoms, where m' is the total number of carbon atoms in the alkyl group).
  • perhaloalkyl means, unless otherwise stated, alkyl substituted with (2m'+l) halogen atoms, where m' is the total number of carbon atoms in the alkyl group.
  • perhalo(Cl-C4)alkyl is meant to include trifluoromethyl, pentachloroethyl, 1,1,1- trifluoro-2-bromo-2-chloroethyl and the like.
  • acyl refers to those groups derived from an organic acid by removal of the hydroxy portion of the acid. Accordingly, acyl is meant to include, for example, acetyl, propionyl, butyryl, decanoyl, pivaloyl, benzoyl and the like.
  • aryl means, unless otherwise stated, a polyunsaturated, typically aromatic, hydrocarbon substituent which can be a single ring or multiple rings (up to three rings) which are fused together or linked covalently.
  • Non-limiting examples of aryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl and 1,2,3,4-tetrahydronaphthalene.
  • heteroaryl refers to aryl groups (or rings) that contain from zero to four heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatom are optionally quaternized.
  • heteroaryl group can be attached to the remainder of the molecule through a heteroatom.
  • heteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4- imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3- thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxal
  • aryl when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above.
  • arylalkyl is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(l-naphthyloxy)propyl, and the like).
  • R', R" and R'" each independently refer to hydrogen, unsubstituted (Cl-C8)alkyl and heteroalkyl, unsubstituted aryl, aryl substituted with one to three halogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(Cl- C4)alkyl groups.
  • R' and R" When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6- or 7-membered ring.
  • -NR'R is meant to include 1-pyrrolidinyl and 4-morpholinyl.
  • an alkyl or heteroalkyl group will have from zero to three substituents, with those groups having two or fewer substituents being preferred in the invention. More preferably, an alkyl or heteroalkyl radical will be unsubstituted or monosubstituted. Most preferably, an alkyl or heteroalkyl radical will be unsubstituted. From the above discussion of substituents, one of skill in the art will understand that the term "alkyl" is meant to include groups such as trihaloalkyl (e.g., -CF 3 and -CH 2 CF 3 ).
  • aryl group is 1,2,3,4-tetrahydronaphthalene, it may be substituted with a substituted or unsubstituted (C3-C7)spirocycloalkyl group.
  • the (C3- C7)spirocycloalkyl group may be substituted in the same manner as defined herein for "cycloalkyl".
  • an aryl or heteroaryl group will have from zero to three substituents, with those groups having two or fewer substituents being preferred in the invention.
  • an aryl or heteroaryl group will be unsubstituted or
  • an aryl or heteroaryl group will be unsubstituted.
  • Preferred substituents for aryl and heteroaryl groups are selected from: halogen, -OR', -OC(0)R', -NR'R", -SR', -R', -CN, -N0 2 , -C0 2 R', -CONR'R", -C(0)R',-OC(0)NR'R", - NR"C(0)R', -S(0)R', -S0 2 R', -S0 2 NR'R", -NR"S0 2 R, -N 3 , -CH(Ph) 2 , perfluoro(Cl- C4)alkoxy and perfluoro(Cl-C4)alkyl, where R' and R" are as defined above.
  • substituents are selected from: halogen, -OR', -OC(0)R', -NR'R", -R', -CN, -N0 2 , - C0 2 R', -CONR'R", -NR"C(0)R', -S0 2 R', -S0 2 NR'R", -NR"S0 2 R, perfluoro(Cl-C4)alkoxy and perfluoro(Cl-C4)alkyl.
  • the substituent -C0 2 H includes bioisosteric replacements therefor; see, e.g., The Practice of Medicinal Chemistry; Wermuth, C. G., Ed.; Academic Press: New York, 1996; p. 203.
  • Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T-C(0)-(CH 2 )q-U-, wherein T and U are independently -NH-, -0-, -CH 2 - or a single bond, and q is an integer of from 0 to 2.
  • two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2)r-B-, wherein A and B are independently -CH 2 -, -0-, -NH-, -S-, -S(O)-, -S(0) 2 -, -S(0) 2 NR'- or a single bond, and r is an integer of from 1 to 3.
  • One of the single bonds of the new ring so formed may optionally be replaced with a double bond.
  • two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CH 2 )s- X-(CH 2 )t- -, where s and t are independently integers of from 0 to 3, and X is -0-, -NR'-, -S-, -S(O)-, -S(0) 2 -, or -S(0) 2 NR'-.
  • the substituent R' in -NR'- and -S(0) 2 NR'- is selected from hydrogen or unsubstituted (Cl-C6)alkyl.
  • the rotation blocking group (RBC or RIO) is bulky group that limits rotation of the substituent at the 4' carbon of anthracene ring core sufficient to substantially reduce fluorescent quenching.
  • a proton (H), F, or other small, or small, linear substituents are insufficiently bulky to quench fluorescence, as is the -AsEDT group of the biarsenicals discussed below.
  • Preferred blocking groups are large, bulky, tetrahedral, and/or aryl functional groups, particularly optionally substituted-, optionally hetero- alkyl, optionally substituted-, optionally hetero- alkenyl, optionally substituted-, optionally hetero- alkynyl, optionally substituted-, optionally hetero-aryl, or optionally substituted-, optionally hetero- alkoxy.
  • the blocking group quench the fluorescence of the compound sufficiently so that when the arcenic atom binds the thiols of the binding target, a practically detectable increase or shift in fluorescence results.
  • the idea is that when the metabolite binds, there is more bulk about the arsenic carbon bond. If there is a substituent in the 475' position, the newly formed ring system will be very bulky and its free rotation will be prevented by the presence of that blocking group. When you limit free rotation, you decrease the efficiency of the quenching process, effectively unquenching the fluorescnce.
  • the target molecule when bound to the compound though a pair of thiols, such as a dicysteine, similarly inhibits rotation at the 5' carbon (note that the 4' and 5' positions can be reversed, depending on the tautomer) of anthracene ring core sufficient to substantially reduce fluorescent quenching.
  • the 5' arsenic atom is typically protected with an arsenic protecting group (Rl 1) displaced by reaction of the arsenic with the thiols of the target molecule.
  • This protecting group should be fluorescent quenching (should not limit free rotation), and is hence, preferably a relatively small, non-bulky group like carbonyl or dihydroxyl, and particulary, small dithiols like 1,2 ethanedithiol. What is important is that target binding provides a detectable increase or shift in fluorescence compared with the protecting group (e.g. EDT)- bound As.
  • the protecting groups may be used to protect the arsenical molecule from reacting with low affinity sites. Dithiol protecting groups may form a five- or six-membered ring with the arsenic. Vicinal dithiols that form rings, such as 5-membered rings, are preferable.
  • Dithiols that contain rings may increase the affinity of the dithiol to the arsenic by organizing the two thiol groups to be in a cis-conformation ready to form an additional ring with the arsenic.
  • Examples of dithiol rings are 1,2 benzenedithiol and 1,2-cyclohexanedithiol.
  • the arsenic is bonded to a dithiol, such as 1,2-ethanedithiol (EDT).
  • a dithiol such as 1,2-ethanedithiol (EDT).
  • the subject compounds encompass the core structures of a wide variety of anthracene-based fluorescent molecules including fluorescein, rhodamine, eosin, phenazine, phenoxazine, phenothiazine, thioxanthene, acridine, and cores and derivatives thereof.
  • anthracene-based fluorescent molecules including fluorescein, rhodamine, eosin, phenazine, phenoxazine, phenothiazine, thioxanthene, acridine, and cores and derivatives thereof.
  • anthracene cores are commercially available or readily synthesized by those skilled in the art. For example, Sigma- Aldrich lists dozens of fluoresceins and derivatives:
  • the subject compounds encompass monoarsenical forms of the biarsenical compounds disclosed in US7,776,999, US7,524,972, US7, 138,503, US6,900,304,
  • the anthracene is initially selected for its optical properties, and may be optimized according to the following criteria: (1) ability to form a fluorescent conjugate with a single molecule of TSH2; (2) brightness of the monoarsenical- TSH2 conjugate; (3) wavelength of absorption ( abs) and the wavelength of emission ( ems); (4) resistance to photobleaching; and (5) rate of conjugation with TSH2.
  • the compounds encompass:
  • Rl is 0; Rl and R2 are C;
  • R3 is 2-carboxyphenyl
  • R4 and R5 are 0, S, N;
  • R4 is amine; R6 and R7 are H; and
  • R5 is amine; R8 and R9 are H.
  • R6 and R7 are H;
  • R8 and R9 are H
  • eosin-based compounds wherein: acridine-based compounds wherein:
  • Rl is 0; Rl is N;
  • R2 is C; R2 is C;
  • R3 is 2-carboxyphenyl; R3 is H or C;
  • R4 is hydroxyl; R4 and R5 are 0, S, or N;
  • R5 is carbonyl
  • R6 and R7 are H
  • R6 and R7 are Br or nitro (-N0 2 ); and R8 and R9 are H.
  • R8 and R9 are H.
  • phenazine -based compounds wherein: acridine yellow (2,7-Dimethylacridine-3,6-
  • Rl and R2 are N; diamine)-based compounds wherein:
  • R3 is H or methyl; Rl is N;
  • R4 and R5 are 0, S, or N; R2 is C;
  • R6 and R7 are H; and R3 is H;
  • R8 and R9 are H R4 and R5 are NH 2 ;
  • R6 and R7 are CH 3 ;
  • R8 and R9 are H phenoxazine -based compounds wherein: acridine orange ( ⁇ , ⁇ , ⁇ ', ⁇ '-
  • Rl is 0; Tetramethylacridine-3,6-diamine)-based
  • R2 is N; compounds wherein:
  • R3 is H or methyl; Rl is N;
  • R4 is hydroxyl; R2 is C;
  • R6 and R7 are H; and R4 and R5 are N(CH 3 ) 2 ;
  • R8 and R9 are H.
  • R6 and R7 are H;
  • R8 and R9 are H. [077] An exemplary synthetic scheme and exemplary compounds 1-4 are shown below.
  • the invention encompasses tautomers, anhydrides and salts of the subject compounds.
  • tautomers anhydrides and salts of the subject compounds.
  • anhydrides for example, a generic 4'-5' tautomer is shown here:
  • the invention also encompasses method of making the subject compounds and conjugates, and methods of using the subject compounds comprising the step of: (a) contacting the compound with the target molecule wherein the arsenic reacts with the thiols, and particularly, a dicysteine.
  • the invention provides a method of diagnosing a trypanasomatid infection in a patient by detecting a dicysteine trypanathione, comprising the steps of: (a) contacting a sample of the patient with a subject compound; and (b) detecting the increase or shift, particularly a red-shift, in fluorescence as an indication of the presence of the trypanathione and the trypanasomatid infection.
  • the invention provides a method of diagnosing a trypanasomatid infection in a patient by detecting a dicysteine trypanathione, comprising the steps of: (a) contacting a sample of the patient with an arsenical fluorescent dye that exhibits a detectable increase or shift in fluorescence when the arsenic of the dye reacts with the cysteines of the trypanathione; and (b) detecting the increase or shift in fluorescence as an indication of the presence of the trypanathione and the trypanasomatid infection.
  • TSH 2 Trypanosomal parasites regulate the oxidative environment inside their cells using a unique redox pair, the metabolite trypanothione (TSH 2 ,) and the enzyme trypanothione reductase (TyR).
  • TSH 2 was first described as the potential target for arsenical therapeutics used to treat these infections. This pathway is orthogonal to that of glutathione and glutathione reductase (GR) found in the human patient or other mammals. Since the arsenical therapeutics have such a high affinity for the sulfur atoms in TSH 2 , we hypothesized that arsenical dyes could be sensitive and selective sensors for the detection of trypanosomal parasites.
  • FlAsH-EDT 2 was virtually non-fluorescent, but upon introduction of TSH 2 , the fluorescence intensity rose. After approximately 10 minutes, the reaction reached saturation. Using the normalized fluorescence measurements, we found that FlAsH-EDT 2 had 0.4% of the fluorescence of the FlAsH-(TSH 2 ) 2 conjugate. The excitation and emission maxima for the conjugate were 505 nm and 527 nm respectively. This increase in fluorescence is readily detectable without a fluorimeter using a simple handheld black light such as those used at airport security checkpoints. This detection method could be implemented easily in a low technology, resource poor setting. We examined the limits of detection of the metabolite with FlAsH-EDT 2 .
  • the field of the invention is arsenical fluorescent agents and diagnostic assays.
  • NTDs Neglected Tropical Diseases
  • Chagas' disease African Trypanosomiasis or Sleeping Sickness
  • Leishmaniasis a group of infections that most commonly plague those who are extremely poor and live in remote rural areas, urban slums, and places of political conflict. Because they adversely affect child development, pregnancy, and worker productivity, NTDs are a major reason why many in developing nations cannot escape extreme poverty. Three such diseases are caused by trypanosomal parasites: Chagas' disease, African Trypanosomiasis or Sleeping Sickness, and Leishmaniasis.
  • the invention provides methods and compositions for labeling dithiol-containing analytes.
  • the invention provides a substituted, optionally hydro-, optionally hetero-, monoarsenical anthracene compound comprising a 4' rotation blocking group and a 5 ' arsenic, and that exhibits a detectable increase or shift in fluorescence when the arsenic reacts with two thiols of a rotation-blocking binding target molecule, the compound having the structure I:
  • R l is O, S, NRa, or CRa 2 ;
  • R2 is C or N
  • R3 is optionally substituted-, optionally hetero- alkyl, optionally substituted-, optionally hetero- alkenyl, optionally substituted-, optionally hetero- alkynyl, optionally substituted-, optionally hetero-aryl, or optionally substituted-, optionally hetero- alkoxy;
  • R4 and R5 are independently carbonyl, carbonothioyl, NRa 2 , ORa, or S R ;
  • R6 and R7 are Ra, halide, ORa, NRa 2 , ORa, SRa, or nitro (-N0 2 );
  • R8 and R9 are Ra, halide, ORa, NRa 2 , ORa, SRa, or nitro (-N0 2 );
  • R I O is a rotational blocking group
  • R l 1 is an arsenic protecting group displaced by reaction of the arsenic with the thiols of the target molecule;
  • Ra groups are independently H, optionally substituted-, optionally hetero- alkyl, optionally substituted-, optionally hetero- alkenyl, optionally substituted-, optionally hetero-
  • R l 1 is carbonyl, 1 ,2 ethanedithiol, or dihydroxyl
  • the rotation blocking group is an optionally substituted-, optionally hetero- alkyl, optionally substituted-, optionally hetero- alkenyl, optionally substituted-, optionally hetero- alkynyl, optionally substituted-, optionally hetero-aryl, or optionally substituted-, optionally hetero- alkoxy
  • the anthracene compound is a fluoresein, rhodamine, eosin, phenazine, phenoxazine, phenothiazine, thioxanthene, acridine, or a core or derivative thereof.
  • the invention provides a substituted, optionally hydro-, optionally hetero-, monoarsenical anthracene compound comprising a 4' rotation- blocking group and a 5 ' arsenic, and that exhibits a detectable increase or shift in
  • AC is the anthracene core
  • RB is the rotation blocking group
  • TM is the target molecule
  • the invention also provide methods of making and using the subject compounds, including a method of using a subject compound comprising the step of: contacting the compound with the target molecule wherein the arsenic reacts with the thiols, and in particular, a method of diagnosing a trypanasomatid infection in a patient by detecting a dicysteine trypanathione, comprising the steps of: (a) contacting a sample of the patient with
  • the invention also provides a method of diagnosing a trypanasomatid infection in a patient by detecting a dicysteine trypanathione, comprising the steps of: (a) contacting a sample of the patient with an arsenical fluorescent dye that exhibits a detectable increase or shift in fluorescence when the arsenic of the dye reacts with the cysteines of the
  • trypanathione and (b) detecting the increase or shift in fluorescence as an indication of the presence of the trypanathione and the trypanasomatid infection.
  • the subject monoarsenical dyes have a significant advantage over diarsenical dyes in diagnoses, in that they form a brightly fluorescent complex upon binding of a dicysteine motif, whereas he 1 : 1 complex with prior diarsenical dyes is only weakly fluorescent. This in not a flaw of the diarsenicals, but reflect their distinct design criteria. Biarsenicals are used in excess to track a small concentration of proteins as they are trafficked through cells, and hence requireaki selectivity and high binding affinity afforded by the tetradentate binding capacity of the bisarsenical. Essentially, the biarsenticals were engineered as a small molecule equivalent of GFP to monitor protein trafficking.
  • the invention provides a substituted, optionally hydro-, optionally hetero-, monoarsenical anthracene compound comprising a 4' rotation-blocking group and a 5 ' arsenic, and that exhibits a detectable increase or shift in fluorescence when the arsenic reacts with two thiols of a rotation-blocking binding target molecule forming a conjugate having the general structure II:
  • AC is the anthracene core
  • RB is the rotation blocking group
  • TM is the target molecule
  • the invention provides a substituted, optionally hydro-, optionally hetero-, monoarsenical anthracene compound comprising a 4' rotation blocking group and a 5' arsenic, and that exhibits a detectable increase or shift in fluorescence when the arsenic reacts with two thiols of a rotation-blocking binding target molecule, the compound having the structure I:
  • Rl is O, S, NRa, or CR 2 ;
  • R2 is C or N
  • R3 is optionally substituted-, optionally hetero- alkyl, optionally substituted-, optionally hetero- alkenyl, optionally substituted-, optionally hetero- alkynyl, optionally substituted-, optionally hetero-aryl, or optionally substituted-, optionally hetero- alkoxy;
  • R4 and R5 are independently carbonyl, carbonothioyl, NRa 2 , ORa, or SRa ;
  • R6 and R7 are Rot, halide, ORa, NRa 2 , ORa, SRa, or nitro (-N0 2 );
  • R8 and R9 are Ra, halide, ORa, NRa 2 , ORa, SRa, or nitro (-N0 2 );
  • R 10 is a rotational blocking group
  • R l 1 is an arsenic protecting group displaced by reaction of the arsenic with the thiols of the target molecule;
  • R groups are independently H, optionally substituted-, optionally hetero- alkyl, optionally substituted-, optionally hetero- alkenyl, optionally substituted-, optionally hetero- alkynyl, optionally substituted-, optionally hetero-aryl, or optionally substituted-, optionally hetero- alkoxy;
  • genuses are recited as shorthand for a recitation of all members of the genus; for example, the recitation of (C 1 -C3) alkyl is shorthand for a recitation of all C 1 -C3 alkyls: methyl, ethyl and propyl, including isomers thereof.
  • heteroatom as used herein generally means any atom other than carbon, hydrogen or oxygen.
  • Preferred heteroatoms include oxygen (O), phosphorus (P), sulfur (S), nitrogen (N), silicon (S), arsenic (As), selenium (Se), and halogens
  • preferred heteroatom functional groups are haloformyl, hydroxyl, aldehyde, amine, azo, carboxyl, cyanyl, thocyanyl, carbonyl, halo, hydroperoxyl, imine, aldimine, isocyanide, iscyante, nitrate, nitrile, nitrite, nitro, nitroso, phosphate, phosphono, sulfide, sulfonyl, sulfo, and sulfhydryl.
  • alkyl by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which is fully saturated, having the number of carbon atoms designated (i.e. C 1 -C8 means one to eight carbons).
  • alkyl groups include methyl, ethyl, n-propyl, isopropyl, n- butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl , n-octyl and the l ike.
  • alkenyl by itself or as part of another substituent, means a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be mono- or polyunsaturated, having the number of carbon atoms designated (i.e. C2-C8 means two to
  • alkenyl groups include vinyl, 2- propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-( l ,4-pentadienyl) and higher homologs and isomers thereof.
  • alkynyl by itself or as part of another substituent, means a straight or branched chain hydrocarbon radical, or combination thereof, which may be mono- or polyunsaturated, having the number of carbon atoms designated (i.e. C2-C8 means two to eight carbons) and one or more triple bonds.
  • alkynyl groups include ethynyl, 1 - and 3-propynyl, 3-butynyl and higher homologs and isomers thereof.
  • alkylene by itself or as part of another substituent means a divalent radical derived from alkyl, as exemplified by -CH2-CH2-CH2-CH2-.
  • an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the invention.
  • a "lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
  • alkoxy alkylamino and “alkylthio” (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively.
  • heteroalkyl by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and from one to three heteroatoms selected from the group consisting of O, N, Si and S, wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
  • the heteroatom(s) O, N and S may be placed at any interior position of the heteroalkyl group.
  • the heteroatom Si may be placed at any position of the heteroalkyl group, including the position at which the alkyl group is attached to the remainder of the molecule.
  • Up to two heteroatoms may be consecutive, such as, for example, -CH 2 -NH-OCH 3 and -CH 2 -0-Si(CH 3 ) 3 .
  • heteroalkylene by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified by -CH2-CH2-S-CH2-CH2- and - CH2-S-CH2-CH2-NH-CH2-.
  • heteroatoms can also occupy either or both of the chain termini (e.g., alkyieneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like).
  • chain termini e.g., alkyieneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like.
  • alkylene and heteroalkylene linking groups no orientation of the linking group is implied.
  • cycloalkyl and heterocycloalkyl represent, unless otherwise stated, cyclic versions of “alkyl " and “heteroalkyl”, respectively. Accordingly, a cycloalkyl group has the number of carbon atoms designated (i.e., C3-C8 means three to eight carbons) and may also have one or two double bonds.
  • a heterocycloalkyl group consists of the number of carbon atoms designated and from one to three heteroatoms selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
  • heterocycloalkyl a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule.
  • cycloalkyl include cyclopentyl, cyclohexyl, 1 -cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like.
  • heterocycloalkyl include l -( l ,2,5 ,6-tetrahydropyrid- yl), 1 -piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,
  • halo and “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
  • terms such as “haloalkyl,” are meant to include alkyl substituted with halogen atoms, which can be the same or different, in a number ranging from one to (2m'+ l ), where m' is the total number of carbon atoms in the alkyl group.
  • halo(C l -C4)alkyl is mean to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
  • haloalkyl includes monohaloalkyl (alkyl substituted with one halogen atom) and polyhaloalkyl (alkyl substituted with halogen atoms in a number ranging from two to (2m'+l ) halogen atoms, where m' is the total number of carbon atoms in the alkyl group).
  • perhaloalkyl means, unless otherwise stated, alkyl substituted with (2m'+ l ) halogen atoms, where m' is the total number of carbon atoms in the alkyl group.
  • perhalo(C l -C4)alkyl is meant to include trifluoromethyl, pentachloroethyl, 1 , 1 , 1 - trifluoro-2-bromo-2-chloroethyl and the like.
  • acyl refers to those groups derived from an organic acid by removal of the hydroxy portion of the acid. Accordingly, acyl is meant to include, for example, acetyl, propionyl, butyryl, decanoyl, pivaloyl, benzoyl and the like.
  • aryl means, unless otherwise stated, a polyunsaturated, typically aromatic, hydrocarbon substituent which can be a single ring or multiple rings (up to three rings) which are fused together or linked covalently.
  • aryl groups include phenyl, 1 -naphthyl, 2-naphthyl, 4-biphenyl and 1 ,2,3,4-tetrahydronaphthalene.
  • heteroaryl refers to aryl groups (or rings) that contain from zero to four heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatom are optionally quaternized.
  • a heteroaryl group can be attached to the remainder of the molecule through a heteroatom.
  • heteroaryl groups include 1 -pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4- imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3- thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1 -isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3- quinolyl
  • aryl when used in combination with other terms (e.g. , aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above.
  • arylalkyl is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-( l -naphthyloxy)propyl, and the like).
  • alkyl group e.g., benzyl, phenethyl, pyridylmethyl and the like
  • an oxygen atom e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-( l
  • R', R" and R'" each independently refer to hydrogen, unsubstituted (C l -C8)alkyl and heteroalkyl, unsubstituted aryl, aryl substituted with one to three halogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(C l - C4)alkyl groups.
  • R' and R" When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6- or 7-membered ring.
  • -NR'R is meant to include 1 -pyrrolidinyl and 4-morpholinyl .
  • an alkyl or heteroalkyl group will have from zero to three substituents, with those groups having two or fewer substituents
  • alkyl or heteroalkyl radical will be unsubstituted or monosubstituted. Most preferably, an alkyl or heteroalkyl radical will be unsubstituted. From the above discussion of substituents, one of skill in the art will understand that the term "alkyl” is meant to include groups such as trihaloalkyl (e.g., -CF 3 and -CH 2 CF 3 ).
  • aryl group is 1 ,2,3,4-tetrahydronaphthalene, it may be substituted with a substituted or unsubstituted (C3-C7)spirocycloalkyl group.
  • the (C3- C7)spirocycloalkyl group may be substituted in the same manner as defined herein for "cycloalkyl".
  • an aryl or heteroaryl group will have from zero to three substituents, with those groups having two or fewer substituents being preferred in the invention.
  • an aryl or heteroaryl group will be unsubstituted or
  • an aryl or heteroaryl group will be unsubstituted.
  • Preferred substituents for aryl and heteroaryl groups are selected from: halogen, -OR', -OC(0)R', -NR'R", -SR', -R', -CN, -N0 2 , -C0 2 R', -CONR'R", -C(0)R',-OC(0)NR'R", - NR"C(0)R', -S(0)R', -S0 2 R', -S0 2 NR'R", -NR"S0 2 R, -N 3 , -CH(Ph) 2 , perfluoro(C l - C4)alkoxy and perfluoro(C l -C4)alkyl, where R' and R" are as defined above.
  • substituents are selected from: halogen, -OR', -OC(0)R', -NR'R", -R', -CN, -N0 2 , - C0 2 R', -CONR'R", -NR"C(0)R', -S0 2 R', -S0 2 NR'R", -NR"S0 2 R, perfluoro(C l -C4)alkoxy and perfluoro(C l -C4)alkyl.
  • Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T-C(0)-(CH2)q-U-, wherein T and U are independently -NH-, -0-, -CH 2 - or a single bond, and q is an integer of from 0 to 2.
  • two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2)r-B-, wherein A and B are independently -CH 2 -, -0-, -NH-, -S-, -S(O)-, -S(0) 2 -, -S(0) 2 NR'- or a single bond, and r is an integer of from 1 to 3.
  • One of the single bonds of the new ring so formed may optionally be replaced with a double bond.
  • two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CH 2 )s- X-(CH 2 )t- -, where s and t are independently integers of from 0 to 3, and X is -0-, -NR'-, -S-, -S(O)-, -S(0) 2 -, or -S(0) 2 NR'-.
  • the substituent R' in -NR'- and -S(0) 2 NR'- is selected from hydrogen or unsubstituted (C l -C6)alkyl.
  • the rotation blocking group (RBC or R 10) is bulky group that limits rotation of the substituent at the 4' carbon of anthracene ring core sufficient to substantially reduce fluorescent quenching.
  • a proton (H), F, or other small , or small, linear substituents are insufficiently bulky to quench fluorescence, as is the -AsEDT group of the biarsenicals discussed below.
  • Preferred blocking groups are large, bulky, tetrahedral , and/or aryl functional groups, particularly optionally substituted-, optionally hetero- alkyl, optionally substituted-, optionally hetero- alkenyl, optionally substituted-, optionally hetero- alkynyl, optionally substituted-, optionally hetero-aryl, or optionally substituted-, optionally hetero- alkoxy.
  • the blocking group quench the fluorescence of the compound sufficiently so that when the arcenic atom binds the thiols of the binding target, a practically detectable increase or shift in fluorescence results.
  • the idea is that when the metabolite binds, there is more bulk about the arsenic carbon bond. If there is a substituent in the 475' position, the newly formed ring system wil l be very bulky and its free rotation will be prevented by the presence of that blocking group. When you limit free rotation, you decrease the efficiency of the quenching process, effectively unquenching the fluorescnce.
  • the target molecule quench the fluorescence of the compound sufficiently so that when the arcenic atom binds the thiols of the binding target, a practically detectable increase or shift in fluorescence results.
  • the 5' arsenic atom is typically protected with an arsenic protecting group (R l 1 ) displaced by reaction of the arsenic with the thiols of the target molecule.
  • This protecting group should be fluorescent quenching (should not limit free rotation), and is hence, preferably a relatively small, non-bulky group like carbonyl or dihydroxyl, and particulary, small dithiols like 1 ,2 ethanedithiol. What is important is that target binding provides a detectable increase or shift in fluorescence compared with the protecting group (e.g. EDT)- bound As.
  • the protecting groups may be used to protect the arsenical molecule from reacting with low affinity sites. Dithiol protecting groups may form a five- or six-membered ring with the arsenic. Vicinal dithiols that form rings, such as 5-membered rings, are preferable.
  • Dithiols that contain rings may increase the affinity of the dithiol to the arsenic by organizing the two thiol groups to be in a cis-conformation ready to form an additional ring with the arsenic.
  • Examples of dithiol rings are 1 ,2 benzenedithiol and 1 ,2-cyclohexanedithiol.
  • the arsenic is bonded to a dithiol, such as 1 ,2-ethanedithiol (EDT).
  • a dithiol such as 1 ,2-ethanedithiol (EDT).
  • the subject compounds encompass the core structures of a wide variety of anthracene-based fluorescent molecules including fluorescein, rhodamine, eosin, phenazine, phenoxazine, phenothiazine, thioxanthene, acridine, and cores and derivatives thereof.
  • anthracene cores are commercial ly avai lable or readily synthesized by those skilled in the art. For example, Sigma-Ald ich l ists dozens of fluoresceins and derivatives:
  • the anthracene is initially selected for its optical properties, and may be optimized according to the following criteria: ( 1 ) ability to form a fluorescent conjugate with a single molecule of TSH2; (2) brightness of the monoarsenical- TSH2 conjugate; (3) wavelength of absorption (?oibs) and the wavelength of emission ( ems); (4) resistance to photobleaching; and (5) rate of conjugation with TSH2.
  • the compounds encompass:
  • R3 is 2-carboxyphenyl
  • R4 and R5 are 0, S, N;
  • R4 is amine; R6 and R7 are H ; and
  • R5 is amine; R8 and R9 are H.
  • R6 and R7 are H;
  • R8 and R9 are H
  • eosin-based compounds wherein: acridine-based compounds wherein:
  • Rl is O; R l is N;
  • R2 is C; R2 is C;
  • R3 is 2-carboxyphenyl; R3 is H or C;
  • R4 is hydroxyl; R4 and R5 are 0, S, or N;
  • R5 is carbonyl
  • R6 and R7 are H
  • R6 and R7 are Br or nitro (-N0 2 ); and R8 and R9 are H.
  • R8 and R9 are H.
  • phenazine -based compounds wherein: acridine yellow (2,7-Dimethylacridine-3,6-
  • Rl and R2 are N; diamine)-based compounds wherein :
  • R4 and R5 are 0, S, or N; R2 is C;
  • R6 and R7 are H; and R3 is H;
  • R8 and R9 are H R4 and R5 are NH 2 ;
  • R6 and R7 are CH3;
  • R8 and R9 are H phenoxazine -based compounds wherein: acridine orange ( ⁇ , ⁇ , ⁇ ', ⁇ '-
  • Rl is O; Tetramethylacridine-3,6-diamine)-based
  • R2 is N; compounds wherein :
  • R4 is hydroxyl; R2 is C;
  • R6 and R7 are H; and R4 and R5 are N(CH 3 ) 2 ;
  • R8 and R9 are H.
  • R6 and R 7 are H ;
  • R8 and R9 are H .
  • the invention encompasses tautomers, anhydrides and salts of the subject compounds.
  • a generic 4' -5 ' tautomer is shown here:
  • the invention also encompasses method of mak ing the subject compounds and conjugates, and methods of using the subject compounds comprising the step of: (a) contacting the compound with the target molecule wherein the arsenic reacts with the thiols, and particularly, a dicysteine.
  • the invention provides a method of diagnosing a trypanasomatid infection in a patient by detecting a dicysteine trypanathione, comprising the steps of: (a) contacting a sample of the patient with a subject compound; and (b) detecting the increase or shi ft, particularly a red-shift, in fluorescence as an indication of the presence of the trypanathione and the trypanasomatid infection.
  • the invention provides a method of diagnosing a trypanasomatid infection in a patient by detecting a dicysteine trypanathione, comprising the steps of: (a) contacting a sample of the patient with an arsenical fl uorescent dye that exhibits a detectable increase or shi ft in fluorescence when the arsenic of the dye reacts with the cysteines of the trypanathione; and (b) detecting the i ncrease or shi ft in fluorescence as an
  • TSH 2 Trypanosomal parasites regulate the oxidative environment inside their cells using a unique redox pair, the metabolite trypanothione (TS H2,) and the enzyme trypanothione reductase (TyR).
  • TSH 2 was first described as the potential target for arsenical therapeutics used to treat these infections. This pathway is orthogonal to that of glutathione and glutathione reductase (GR) found in the human patient or other mammals. S ince the arsenical therapeutics have such a high affinity for the sulfur atoms in TSH 2 , we hypothesized that arsenical dyes could be sensitive and selective sensors for the detection of trypanosomal parasites.
  • FIASH-EDT2 was virtually non-fluorescent, but upon introduction of TS H2, the fluorescence intensity rose. After approximately 1 0 minutes, the reaction reached saturation. Using the normalized fluorescence measurements, we found that FIASH-EDT2 had 0.4% of the fluorescence of the FlAsH-(TSH2) 2 conjugate. The excitation and emission maxima for the conjugate were 505 nm and 527 nm respectively. This increase in fluorescence is readily detectable without a fluorimeter using a simple handheld black l ight such as those used at airport security checkpoints. This detection method could be implemented easily in a low technology, resource poor setting. We examined the limits of detection of the metabolite with Fl AsH-EDT 2 .

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  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
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US6008378A (en) * 1997-10-21 1999-12-28 The Regents Of The University Of California Synthetic molecules that specifically react with target sequences
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BRPI0603872B8 (pt) * 2006-08-24 2021-07-27 Fundacao Univ De Brasilia método de diagnóstico e de monitoramento do tratamento de tripanossomíases crônicas, método de diagnóstico e de monitoramento do tratamento da doença de chagas crônica, e kit de diagnóstico para uso no diagnóstico e no monitoramento do tratamento de tripanossomíases crônicas e da doença de chagas

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US20110229929A1 (en) 2011-09-22
US8664009B2 (en) 2014-03-04
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JP5476504B2 (ja) 2014-04-23
CA2788292A1 (en) 2011-09-22
WO2011116401A2 (en) 2011-09-22
JP2013522632A (ja) 2013-06-13
BR112012023671A2 (pt) 2017-10-03
US20140162309A1 (en) 2014-06-12
WO2011116401A3 (en) 2012-01-19

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